Cell-free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer.

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour-enriched tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next-generation sequencing was performed on ascites-derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites-derived tumour cells (n = 5) and archived formalin-fixed paraffin-embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

Rates of paclitaxel hypersensitivity reactions using a modified Markman's infusion protocol as primary prophylaxis.

PURPOSE: Markman's desensitisation protocol allows successful retreatment of patients who have had significant paclitaxel hypersensitivity reactions. We aimed to reduce the risk and severity of paclitaxel hypersensitivity reactions by introducing this protocol as primary prophylaxis. METHODS: We evaluated all patients with a gynaecological malignancy receiving paclitaxel before (December 2018 to September 2019) and after (October 2019 to July 2020) the implementation of a modified Markman's desensitisation protocol. The pre-implementation group received paclitaxel over a gradually up-titrated rate from 60 to 180 ml/h. The post-implementation group received paclitaxel via 3 fixed-dose infusion bags in the first 2 cycles. Rates and severity of paclitaxel hypersensitivity reactions were compared. RESULTS: A total of 426 paclitaxel infusions were administered to 78 patients. The median age was 64 years (range 34-81), and the most common diagnosis was ovarian, fallopian tube and primary peritoneal cancer (67%, n = 52/78). Paclitaxel hypersensitivity reaction rates were similar in the pre-implementation (8%, n = 16/195) and post-implementation groups (9%, n = 20/231; p = 0.87). Most paclitaxel hypersensitivity reactions occurred within 30 min (pre- vs. post-implementation, 88% [n = 14/16] vs. 75% [n = 15/20]; p = 0.45) and were grade 2 in severity (pre- vs. post-implementation, 81% [n = 13/16] vs. 75% [n = 15/20]; p = 0.37). There was one grade 3 paclitaxel hypersensitivity reaction in the pre-implementation group. All patients were successfully rechallenged in the post-implementation group compared to 81% (n = 13/16) in the pre-implementation group (p = 0.43). CONCLUSION: The modified Markman's desensitisation protocol as primary prophylaxis did not reduce the rate or severity of paclitaxel hypersensitivity reactions, although all patients could be successfully rechallenged.

"Worlds. . .[of] Contingent Possibilities": Genderqueer and Trans Adolescents Reading Fan Fiction.

Cultural studies scholars have long been interested in the nexus between people's online activities and their identities. One activity that has drawn attention is reading/writing fan fiction (fictions written by and for fans that build upon the characters and worlds depicted in commercial texts). While fan fiction and its surrounding communities have long been understood as resistant to heteronormativity, previous work exploring the fans who produce and consume fan fiction has largely insisted that most of these fans are adult ciswomen. Little has been written about the experiences of trans and genderqueer fans. To remedy this elision, this article explores two trans and genderqueer individuals' experiences with fan fiction. It closely examines the roles reading, and especially reading fan fiction, has or has not played in their understandings of themselves, their identities, and their places in the world.

Transformative Readings: Harry Potter Fan Fiction, Trans/Queer Reader Response, and J. K. Rowling.

The politics of children's literature and the actors surrounding it have never been more visible than they are now, in the digital age. As one of the first children's series to gain widespread popularity concurrently with the spread of the internet, the Harry Potter septet arrived on the global stage at the perfect moment to develop an avid, connected fandom. But the fandom has laid bare the many conflicting ideologies of the fans themselves and of the actors surrounding the texts. This article examines the contentious issue of gender nonnormativity and its relation to the Harry Potter texts, the queer/trans reading practices and political resistance common to the fandom, and the ongoing disagreements over gender, made visible on social media, between Rowling and the fans of her series. The article discusses the Harry Potter novels' varied and conflicting ideologies; queer/trans readings of the Potter septet, including both invitations and resistances to queer/trans reading by Rowling herself; how gender is queered and queried in and through fan fiction; and finally, the recent hostilities between Rowling and her fans. It concludes by discussing the worsening relationship between Rowling and her fans and highlighting how fans are using their collective power to undermine Rowling's gender politics through fan fiction. By doing so, the article traces the complex politics of the reception of books for young people in the digital age, demonstrating that authors' powerful voices continue to shape readers' responses to texts long after their publication but showing, too, that readers often resist authors' attempts to influence not only their textual interpretations but their politics.

Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.

OBJECTIVE: Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content. METHODS: Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating. RESULTS: The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h. CONCLUSION: cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer.

Connected While Distant: Networking CUREs Across Classrooms to Create Community and Empower Students.

Connections, collaborations, and community are key to the success of individual scientists as well as transformative scientific advances. Intentionally building these components into science, technology, engineering and mathematics (STEM) education can better prepare future generations of researchers. Course-based undergraduate research experiences (CUREs) are a new, fast-growing teaching practice in STEM that expand opportunities for undergraduate students to gain research skills. Because they engage all students in a course in an authentic research experience focused on a relevant scientific problem, CUREs provide an opportunity to foster community among students while promoting critical thinking skills and positively influencing their identities as scientists. Here, we review CUREs in the biological sciences that were developed as multi-institutional networks, and highlight the benefits gained by students and instructors through participation in a CURE network. Throughout, we introduce Squirrel-Net, a network of ecology-focused and field-based CUREs that intentionally create connections among students and instructors. Squirrel-Net CUREs can also be scaffolded into the curriculum to form connections between courses, and are easily transitioned to distance-based delivery. Future assessments of networked CUREs like Squirrel-Net will help elucidate how CURE networks create community and how a cultivated research community impacts students' performance, perceptions of science, and sense of belonging. We hypothesize networked CUREs have the potential to create a broader sense of belonging among students and instructors alike, which could result in better science and more confident scientists.

Carnivore use of avocado orchards across an agricultural-wildland gradient.

Wide-ranging species cannot persist in reserves alone. Consequently, there is growing interest in the conservation value of agricultural lands that separate or buffer natural areas. The value of agricultural lands for wildlife habitat and connectivity varies as a function of the crop type and landscape context, and quantifying these differences will improve our ability to manage these lands more effectively for animals. In southern California, many species are present in avocado orchards, including mammalian carnivores. We examined occupancy of avocado orchards by mammalian carnivores across agricultural-wildland gradients in southern California with motion-activated cameras. More carnivore species were detected with cameras in orchards than in wildland sites, and for bobcats and gray foxes, orchards were associated with higher occupancy rates. Our results demonstrate that agricultural lands have potential to contribute to conservation by providing habitat or facilitating landscape connectivity.

Patient transfer forms enhance key information between nursing homes and emergency department.

The purpose of this study was to determine whether the use of a transfer form increases the availability of essential information needed for patient care and to examine its effect on case resolution time and disposition status. A retrospective review was performed that included patients 65 years old or greater arriving from nursing homes to an academic Emergency Department from January to June 2009. Eighty randomly selected charts were reviewed. Sixteen items were deemed essential based on an expert-based rubric published in 2006. Each case was scored accordingly. Results indicate that the transfer form group had available, on average, 71% of the essential items as compared to 28% for cases without a transfer form (p < 0.001). There was no difference in the case resolution time (p = 0.94) or in disposition status (p = 0.12). In conclusion, essential information for providing emergency department patient care was significantly increased with the use of a transfer form.

Effects of chloride substitution on electromechanical responses in the pulmonary artery of Dahl normotensive and hypertensive rats.

1. We have investigated the in vitro interaction between chloride ions and endothelium as revealed by alterations in vascular contractility and smooth muscle cell membrane potential in isolated pulmonary arteries from Dahl salt-resistant normotensive and salt-sensitive hypertensive rats. 2. Exposure to nitro-l-arginine methyl ester (l-NAME) of tissues from normotensive but not hypertensive rats augmented contractions to cirazoline. While chloride removal did not alter cirazoline-induced contractions, it completely abolished the augmentation by l-NAME in normotensive rats. However, in hypertensive rats, removal of chloride ions significantly attenuated contractions elicited by cirazoline, and l-NAME effectively reversed this inhibition. 3. Methacholine-induced endothelium-dependent relaxations of the same magnitude were evident in both normotensive and hypertensive rats. However, basal cyclic GMP levels were found to be significantly higher (7.8-fold) in blood vessels of normotensive rats compared to hypertensive rats. 4. The resting membrane potential in pulmonary arteries of hypertensive rats (-52.1+/-1.04 mV) revealed a significant hyperpolarisation when compared with that of normotensive rats (-46.4+/-1.58 mV). Cirazoline did not produce a significant depolarisation in blood vessels of either normotensive or hypertensive rats. Perfusion with chloride-free solution resulted in a modest but significant hyperpolarisation (-8.0 mV) in the blood vessels of hypertensive but not in normotensive rats. 5. We conclude that salt-dependent hypertension in Dahl rats is accompanied by functional and biochemical changes in low-pressure blood vessels. These changes can, in part, be attributed to impairment in the basal, but not methacholine-stimulated, release of nitric oxide, and to altered chloride ion handling.

The influence of calcium channel antagonists on isolated human distal radial arteries.

A comparison was made between the efficacies and potencies of calcium channel antagonists on contractions induced by 5-hydroxytryptamine, norepinephrine, or high-potassium buffer in isolated human distal radial arteries. Also, the impact of removal of extracellular calcium ions on contractions induced by 5-hydroxytryptamine or norepinephrine was assessed. Isometric contractions were induced by agonists, and relaxant responses to calcium channel antagonists, nifedipine, diltiazem, verapamil, or mibefradil were examined. 5-hydroxytryptamine-induced contractions when compared with norepinephrine or high-potassium induced contractions were significantly more sensitive to inhibition by nifedipine (pIC50 = 7.53 +/- 0.15; 6.78 +/- 0.12; 6.6 +/- 0.22, respectively, mean +/- SEM). Diltiazem was more effective in producing relaxations when contractions were elicited with 5-hydroxytryptamine (pIC50 = 6.48 +/- 0.84) or norepinephrine (pIC50 = 6.20 +/- 0.21) than with high potassium (pIC50 = 5.43 +/- 0.10). Verapamil was more effective at relaxing arteries contracted with 5-hydroxytryptamine (pIC50 = 6.09 +/- 0.19) or norepinephrine (pIC50 = 6.00 +/- 0.17) than with high potassium (pIC50 = 5.60 +/- 0.16). Mibefradil was not very effective in producing relaxations. The studies revealed that removal of extracellular calcium significantly attenuated contractions produced by 5-hydroxytryptamine (-67.7 +/- 6.3%) and norepinephrine (-89 +/- 1.5%). In conclusion, our data indicates that nifedipine was the most effective drug in producing relaxations; also, contractions produced by 5-hydroxytryptamine and norepinephrine were found to be critically dependent on the presence of extracellular calcium.

Effect of nitric oxide synthase inhibitor N(omega) nitro-L-arginine methyl ester on relaxant responses to calcium channel antagonists in isolated aortic rings from Dahl normotensive and hypertensive rats.

Differences exist between the pharmacological actions of calcium channel antagonists in blood vessels from hypertensive versus normotensive animals. In this investigation, we have examined the impact of nitric oxide synthase inhibitor N(omega) nitro-L-arginine methyl ester (L-NAME) on relaxant responses produced by the calcium channel antagonists (nifedipine, diltiazem, and mibefradil) in isolated aortic rings from Dahl salt-resistant normotensive (SRN) and salt-sensitive hypertensive (SSH) rats on a 4% salt diet. Morphological examination of the aortic rings revealed significantly larger lumen area, smooth muscle wall thickness, and perimeter in vessels of SSH rats versus SRN rats. Rank order potency for the antagonists was nifedipine > mibefradil > or = diltiazem in aortic rings from SRN rats, but mibefradil was found to be the most efficacious. The rank order potency for the antagonists in aortic rings from SSH rats was nifedipine > diltiazem > mibefradil, although all three drugs showed similar efficacy. The presence of L-NAME attenuated relaxations elicited by the antagonists in aortic rings from SRN rats. Treatment of tissues with L-NAME significantly reduced maximal response and decreased pIC(50). The presence of L-NAME had no effects on concentration-response curves to nifedipine and diltiazem in aortic rings from SSH rats, but it significantly attenuated relaxant responses of mibefradil. Our current results support the view that these calcium channel antagonists produce relaxations by mechanisms that are sensitive and insensitive to L-NAME. Moreover, the component insensitive to L-NAME was lacking in tissues from SSH rats for nifedipine and diltiazem but not mibefradil.

Comparative study of functional responses and morphometric state of distal radial arteries in male and female.

BACKGROUND: Differences can exist in terms of physiology and morphology of blood vessels on the basis of gender. Radial artery is now considered to be the second choice for coronary artery bypass grafting. However, there is a lack of comparative studies on the function and morphometery of radial arteries in female and male patients. METHODS: Radial arteries from 9 female and 9 male patients undergoing coronary artery bypass grafting were used to compare the effects of vasoconstrictors, noradrenaline and 5-hydroxytryptamine, as well as the influence of endothelium-dependent (with methacholine) and endothelium-independent (with sodium nitroprusside) relaxations. Furthermore, morphomteric measurements of smooth muscle thickness, lumen perimeter, lumen area, and intima area (including plaque) of distal radial arteries from female and male patients were also made. RESULTS: Radial arteries from female patients when compared to male patients were significantly more sensitive to the actions of noradrenaline, and somewhat more sensitive towards the actions of 5-hydroxytryptamine. However, no significant differences were found between the relaxant effects of methacholine in radial arteries of female and male patients. In contrast, radial arteries from female patients when compared to male patients were significantly less sensitive to the relaxant effects of sodium nitroprusside. Morphometric measurements of blood vessels from female and male patients revealed that vessels obtained from female patients had a smaller lumen area and perimeter than vessels from male patients. In contrast, there were no significant differences between tunica intima area (including plaque area) or smooth muscle thickness in radial arteries of female patients when compared to male patients. However, the radial arteries from female patients had a significantly greater ratio of tunica intima area (including plaque) to lumen area when compared with radial arteries from male patients. CONCLUSIONS: Differences exist between the functional behavior and morphometery of radial arteries of female and male patients. It is possible that postbypass, radial artery graft may show different characteristics in female versus male patients.